ANTIPLASMODIAL ACTIVITIES OF THE STEM BARK EXTRACT OF ARTOCARPUS ALTILIS FORSBERG

Background: The potential of Artocarpus altilis stem bark as a safe antimalarial agent, and the identification of its antimalarial constituents was explored. Materials and Methods: The air-dried stem bark was extracted with 70% ethanol, filtered and concentrated in vacuo to obtain the extract (EE). The extract was successively partitioned to give n-hexane (AAH), dichloromethane (AAD), ethyl acetate (AAE) n-butanol (AAB) and aqueous (AAQ) fractions respectively after determining the acute toxicity using Lorke’s method. These were each evaluated for chemosuppressive antimalarial activities (0-200mg/kg) against chloroquine-sensitive Plasmodium berghei-berghei-infected albino mice. Normal saline and chloroquine, 10 mg/kg were negative and positive control respectively.The survival times and percentage survivors of the mice in both experiments were determined after observation for twenty-eight days post-drug administration. The five (5) column chromatographic (CC) fractions, AAH1, AAH2, AAH3, AAH4 and AAH5 obtained from the most active AAH, were also evaluated for antimalarial activities (0-50mg/kg). Further column purification and repeated PTLC of AAH5 yielded three bands, which were finally subjected to GC-MS analysis. Results: EE gave ED50 and LD50 values of 227.17and >5000 mg/kg while its partitioned fractions gave ED50 values as follows: AAH, 79.14; AAD, 215.59; AAE, 160.46, AAB, .42; and AAQ, 90.85 mg/kg respectively. The primary CC fractions also gave ED50 values as follows: AAH1 21.95; AAH2, 26.96; AAH3, 21.30; AAH4, 20.92 and AAH5, 20.75 mg/kg respectively to identify AAH5 as the putative fraction. GC-MS analysis revealed eleven major compounds (1–11) in the three PTLC bands as the antiplasmodial constituents of the plant. Conclusion: The stem bark of A. altilis is a potential agent in malaria control which is safe for oral use.


Introduction
contain a standard inoculum of 1x10 7 infected erythrocytes in 0.2 mls of diluted blood. This was administered intraperitoneally to Swiss albino mice weighing between 18 and 22g. The parasite was subsequently maintained by serial passaging in mice and by close monitoring of the parasitaemia level (Peters, 1965).

(ii) The four-day suppressive test
The in vivo antimalarial activities of the ethanol extract and fractions were determined using the four-day suppressive test (Peters, 1965). Experimental Swiss albino mice that have been allowed to acclimatize for at least 10 days earlier were inoculated with the Plasmodium parasite and randomized into different groups of five mice each as appropriate for each experiment including the negative (distilled water) and the positive control (CQ, 10 mg/kg). These were administered with the various doses of the extracts or fractions orally 2 hours after inoculation (Do) and repeated daily for the following three days (D1, D2, D3). On the fifth day (i.e., D4), blood was withdrawn from the tail of each mouse and the level of parasitaemia determined. The mice were further observed for 28days, for mortality, from the day of drug administration.

(iii) Average percentage parasitaemia, percentage chemosuppression and median effective doses
The parasitaemia level in each mouse as percentage parasitaemia was determined by counting the number of parasitised (NPE) and unparasitised (NUE) red blood cells for each of ten fields in a blood smear view under the oil immersion objective of a microscope and estimating from the formula:100(NPE)/(NPE + NUE). The average of these, for 5 mice were calculated to give the average percentage parasitaemia per dose (Peters, 1965). The Percentage (%) chemo-suppression for each dose was afterwards calculated from the average percentage parasitaemia using the formula: APU-APT x 100/ APU where APU is average percentage parasitaemia in the untreated mice and APT is average percentage parasitaemia in the treated mice (Peters, 1965). The median effective doses, ED50 and ED90 values, which are measures of the antimalarial activity, are the doses required to reduce parasitaemia in infected mice by 50 and 90 %, respectively and were determined as forecasted from a plot of the percentage chemosuppression against the test dose using a Microsoft Excel 2007 programme.

(iv) Survival Times and Percentage survivors
The mice for each of the above experiments were observed daily for 28 days for mortality from the day of drug administration (Peters, 1965). The average numbers of days for which the mice survived per group were determined and recorded as mean ± SEM. The percentage survivor (PS) was recorded as a percentage of total number of animals per group that gave survival times greater than or equal to the average survival time obtained for the group. It was calculated using the formula: NS /NT X 100 where NS is the number of animals with survival times greater than or equal to the average group survival time and NT is the total number of animals per group.

In vivo Antimalarial Assays of the ethanolic extract of A. altilis
The ethanolic extract was tested for antimalarial activities using the four-day test described in section (ii) above. Briefly, thirty (30) infected mice were randomized into six groups of five (5) each to be administered with doses 25, 50, 100 and 200 mg/kg of ethanolic extract (groups I-IV), chloroquine,10 mg/kg, group V as the positive control and distilled water (group VI as the negative control). These were given orally 2 hours after inoculation (Do) and repeated daily for the next three days (D1, D2, D3). On the fifth day (i.e., D4), the level of parasitaemia was determined for each mouse by withdrawing blood from the tail of each of the animals. The percentage (%) chemo suppression for each dose was afterwards calculated as in section (iii) above. The ED50 and ED90 values were forecasted from the percentage chemosuppression value for each dose using the Microsoft Excel 2007 programme. The mice were further observed for 28 days after drug administration for mortality and the survival times and percentage survivors determined as in Section (iv) above.

Purification of the Stem-bark of A. altilis Extract Pre-column purification
The A. altilis stem bark extract (170 g) was adsorbed on 70-230 mesh silica gel (70.0 g) and air-dried to obtain a free-flowing consistency. This was packed into a sintered Buchner funnel and eluted successively under pressure with nhexane (1500 ml) dichloromethane (2500 ml), ethyl acetate (2500 ml), and n-butanol (300 ml), and water (300 ml). Thin-Layer Chromatography was used to monitor the elution which was exhaustive at each stage before changing the solvents. The resulting fractions were concentrated in vacuo to give the n-hexane, AAH (9.63 g), AAD dichloromethane (18.66 g), AAE ethyl acetate (18.99 g), AAB n-butanol (10.59g), and AAQ aqueous (29.45 g) fractions respectively. The yields were calculated.

In vivo Antimalarial Assays of the partition fractions of A. altilis
The five (5) partition fractions, n-hexane, dichloromethane, ethyl acetate, n-butanol, and aqueous were separately tested for antimalarial activities at doses 12.5, 25.0 and 50 mg/kg using the four-day test described in Section (ii) above. The percentage (%) chemo suppression for each dose was afterwards calculated as in section (iii) above. The ED50 and ED90 values, the survival times and percentage survivors were determined as above in Section (iii) and (iv) above.

In vivo antimalarial assays of the bulked column fractions of the n-hexane fraction
The pooled column fractions AAH1, AAH2, AAH3, AAH4 and AAH5 of the n-hexane partition fraction of A. altilis stem bark were subjected to the in vivo antimalarial four-day suppressive (Peters, 1965), as in Section (ii) above. The percentage (%) chemo suppression for each dose was afterwards calculated as in section (iii) above. The ED50 and ED90 values, the survival times and percentage survivors were determined as above in Section (iii) and (iv)above.

Statistical Analysis
The antimalarial activities of A. altilis extract, partition and bulked column fractions of the most active nhexane fraction were shown by comparing their percentage chemosuppression and ED50, ED90 with those of the positive and negative control and with each other by subjecting the values to statistical analysis using ANOVA followed by Dunnett and Bonferroni t-test as the post-hoc tests. P < 0.05 was considered as significant.

Purification of the most active column fraction AAH5
The most active column fraction AAH5 (2.0 g) was purified to give AAH5 I-IV of which AAH5 III was chosen for further purification. Therefore, 600.0 mg of AAH5 III was adsorbed with 500.0 mg of silica gel and eluted with gradient concentrations of dichloromethane and ethyl acetate on a silica gel column packed with dichloromethane, as follows:

Gas Liquid Chromatographic Analysis
Gas Liquid Chromatographic (GLC) separation was performed on a Gas chromatography (Agilent, USA) hyphenated to a mass spectrophotometer (5957C) with triple axis detector equipped with an auto injector (10 µL syringe) with Helium gas as carrier. All chromatographic separation were performed on a capillary column, specification 19091S-433 HP-5MS, dimensions: 30 m x 250 µm x 0.25 µm, treated with phenyl methylsilox and operated at a constant flow rate of 1.5 mL/min of helium gas with other conditions as follows: EI (ion source temperature), 250 0 C, interface temperature 300 0 C, pressure 16.2 psi, out time 1.8 mins; 1.0ml injector in the split mode with a split ratio 1:50 and an injection temperature of 300 0 C. The oven temperature was held for 5.0 min at an initial temperature of 35 °C and programmed to increase to 150 °C at 4 °C/min, held for 2 min and later increased to 250 °C at 20° C/min and finally held isothermally for 5 minutes, giving a total run time of 47.5 mins. Transfer line temperature was set to 34 °C and post run temperature was to 325 °C for 10 min. The data solution software supplied was used to control the system and acquire the data. The separated constituents were passed to the detector which recorded the emergence of the constituents as peaks with a retention time. The percentage compositions of the compound in the entire sample were computed from the peak areas automatically generated by the machine. The results were recorded as retention time against percentage composition in the original sample.

Gas Chromatographic-Mass Spectrometric (GC-MS) Analysis of A1, A2 and A3
C-MS analysis was performed on A1, AAH5 III 2-3a; A2, AAH5 III 2-3b and A3, AAH5 III 4-5b as stated above. Samples were prepared and injected into the GC-MS machine and the result acquired as peaks with respective retention times.
Data handling was done using GC-MS solution software. The identities of the components were assigned by comparing their retention times with those of the standard spectra from NIS

Ethical Approval
The protocol used for this study was approved by the Board of Postgraduate College, OAU with the Registration Number PHP/H/10/11/1017. Principles of laboratory animal care" (NIH publication No. 85-23, revised 1985) were followed, as well as specific national laws where applicable.

Acute toxicity test
No mortality was observed at >5000mg/kg it means that the extract is orally safe (Lorke, 1983). Keys: Data show the mean ± SEM, n = 5: NC (negative control): Tween 80 in normal saline; CQ = Chloroquine (10 mg/kg). Only values with different superscripts of alphabets within columns are significantly different (p < 0.05, one-way analysis of variance followed by the Student-Newman-Keul's post hoc test).

Discussion and Conclusion
Medicinal plants with claims of antimalarial activities are potential sources of antimalarial agents (Iwalewa et al., 2008). Therefore, research efforts have been directed at verifying such claims by scientists using multifarious approaches such as activity-directed in vivo investigation of plant extracts on rodent parasite infected models in order to establish antimalarial action (Carvalho et. al., 1991). Some plants such as Quassia amara, Quassia undulate, Zingiber officinale, Acacia nilotica, Xylopia aethiopica and Artemisia maciverae have been screened through such model for antimalarial activities (Ajaiyeoba et al., 1999).
Incidence of renal and hepatic toxicity has been recorded with the ingestion of the medicinal herbs particularly at high dose (Pieme et al., 2006), hence the need for evaluation of their safety and efficacy profile (Ogbonnia et al., 2010). The stem-bark of Morinda lucida was reported to be extremely toxic (Ajaiyeoba et al., 2006). Therefore, in this study, toxicity evaluation preceded anti-plasmodial testing. The LD50 values are the usual indices of toxicity potentials of plant extracts and also a means of determining the safe doses to be used for testing the activities of extracts. The LD50 determined for the ethanolic extract of the A. altilis stem-bark was above 5,000mg/kg. This implies that the oral use of A. altilis extract is safe (Lorkes, 1983) and the doses 0-800 mg/kg body weight chosen for the ethanolic extract is justified.
The in vitro and in vivo antimalarial tests are established models for assessing antimalarial activities and both should be performed to justify the antimalarial activities of a plant extract or fraction (Aladesanmi et al, 1988;Simons 2006, Adebajo et al., 2013). The in vitro antimalarial property of A. altilis has been reported (Boyom et al., 2009); in vivo activity was therefore attempted to complement and justify antimalarial efficacy of this plant. P. berghei, a rodent parasite was chosen for the in vivo chemosuppressive activity in the four-day antimalarial test (Peters, 1965). This method was also used by Ebiloma et al., 2011 in the investigation of the antimalarial activity of Morinda lucida (Benth) against erythrocytic stage of mice infected with chloroquine sensitive Plasmodium berghei NK-65.

Antiplasmodial activities
The percentage chemosuppression derived from the percentage parasitaemia is employed as a suitable measure of anti-malarial activity especially in the in vivo experiment (Ebiloma et al., 2011). The dose-dependent percentage chemosuppression of the ethanolic extract up to 200 mg/kg (Table1) was comparable to chloroquine, an established drug used in the treatment of malaria (Aliyu, 2013). This activity of A. altilis may be due to a synergistic combination of some constituents present in the extract. The effect may also likely be better against resistant parasite strains than chloroquine. However, solvent partitioning of the ethanolic extract into different solvents was to profile the chemical constituents according to their polarity. The significantly lower ED50 and ED90 values of n-hexane partition fraction compared to that of ethyl acetate and butanol (Tables 2 and 3) informed its choice as the most active partition fraction.
However, dichloromethane partition fraction gave the lower percentage of chemosuppression at 50 mg/kg dose and higher ED50 values compared to the aqueous partition fractions. The order of activity therefore for the partitioned fractions is AAH>AAB>AAQ>AAE>AAD. All were more active than the ethanol extract. The n-hexane fraction was therefore purified by column chromatography and the five pooled fractions, AAH1, AAH2, AAH3, AAH4 and AAH5 obtained were further subjected to anti-malarial testing.
All the pooled column fractions elicited a dose-dependent percentage chemosuppression (Table 5) but comparable ED50 and ED90 except the AAH2 (Table 6), though were all significantly better in activity than those of the ethanolic extract and n-hexane fraction ( Table 1). The order of activity for the pooled column fractions is AAH1=AAH3=AAH4=AAH5>AAH2. AAH5 was chosen for purification by a further column chromatography.

Survival times and Percentage Survivors (PS)
The n-hexane partition fraction, at 25mg/kg, elicited survival times that was significantly different from that elicited by negative control and comparable to that of chloroquine, the positive control drug (Table 4). Also, at the same dose of 25mg/kg, n-hexane produced significantly (p<0.05) higher survival times in mice than other partition fractions. However, the survival times elicited by all the doses of the AAD partition fraction except that of 100mg/kg were comparable to that of the positive control. All the doses of the ethyl acetate fraction just like the butanol fraction produced lower survival time values which were all comparable to the negative control. For the aqueous fraction, only 50 mg/kg gave values that were comparable (p>0.05) to CQ, others produced values which were significantly lower than CQ. Therefore, the order of activity using the survival time for the partition fraction is AAH>AAD>AAQ>AAB= AAE. The better survival times elicited by the n-hexane partition fraction (AAH) than other fractions further confirm it as the most active partition fraction.
The survival time produced by AAH1 just like AAH3 at 12.5mg/kg dose was significantly (p<0.05) higher than that produced by the positive control. The values were also comparable to that elicited by those of 25 and 50mg/kg doses. Also, the percentage survivor (PS) of 60% given by AAH1 and AAH3 at 12.5mg/kg corroborate it's significantly (p<0.05) higher activity than the positive control with 40% PS. Also, the 80% PS given by 50mg/kg, the highest tested dose for AAH3 attested to its better activity than AAH1 while AAH2 and AAH4 gave comparable survival time values at all the doses tested to the negative control. The AAH5 at 12.5mg/kg gave significantly (p<0.05) higher survival times than the other doses including the negative control. It also showed comparable activities to the positive control drug and a PS of 40% at all its tested doses. Therefore, the order of activity using the survival time for the column bulked fractions is AAH1>AAH3>AAH5>AAH2=AAH4. The significantly high percentage chemosuppression, the low ED50 and ED90 values coupled with the very high survival time of the n-hexane partitioned fraction made it the choice for further purification. The n-hexane extract and purified bulked fractions of K. grandifoliola have been reported to give 91 % chemosuppression in vivo in mice and IC50 values of 1.4 µg/mL (for multiple-drug resistant clone) or 0.84 µg/mL (for Nigerian P. falciparum isolates) (Agbedahunsi et al ., 1998). Also, the n-hexane fraction of H. madagascariensis exhibited the highest suppressive activity of 93.94 % at 40 mg/kg among other fractions (Iwalewa, et al., 2008). Though all the bulked column fractions of the most active n-hexane partition fraction, except AAH2 gave comparable (p>0.05) ED50 and ED90 values, AAH1 and AAH3 gave comparably higher survival times hence suitable candidate fractions for further purification. However, the thin-layer chromatography (TLC) of all the column fractions showed the respective number of visible spots and weight as follows: AAH1 (8 spots, 0.41g), AAH2 (13 spots, 1.21 g), AAH3 (12 spots, 0.29 g), AAH4 (6 spots, 0.66 g), AAH5 (4 spots, 2.60 g). Therefore, further purification works should be concentrated on these bulked column fractions particularly AAH1 and AAH3 with high antiplasmodial activities. However, AAH5 was chosen for further purification because of its weight, the fewer number of TLC spots and the highest survival times (Table 7). It was purified, therefore, by a further column chromatography to give AAH5 I, AAH5 II, AAH5III, AAH5 IV and AAH5 V from which AAH5 III was further subjected to repeated PTLC to give A1 (AAH5 III 2-3 a (16mg), A2 AAH5 III 2-3 b (13mg) and A3 AAH5 III 4-5b (14mg) which were chosen for GC-MS analysis.

Identification of the constituents of the most active PTLC bands isolates after GC-MS
The hyphenated technique of Gas chromatography-mass spectrometry (GC-MS) is a valuable tool in natural product research, assisting in the separation and identification of chemical components of complex volatile mixtures found in petrochemicals and volatile oils of plants in many natural product researches (Li et al., 2009;David et al, 2015), and organic extracts (Kimaru and Nguyen, 2014;Gomathi et al. 2015). It is known for its high-resolution and separations of isomeric structurally similar mono-and sesqui-terpenes that are the main constituents of plant essential oils. In practice, isolation is only mandatory when a component is suspected to be new (Kimaru and Nguyen, 2014). The use of electron ionisation-mass spectrometry (EI-MS) produces distinctive mass spectral fragmentation patterns, which when compared with stored EI spectra in the computer library, may help in identifying the separated components. In hyphenation with GC therefore, using the Rt, MS library and co-injection of pure isolates (reference standards), most peaks of the GC analyses of volatile oils have been conclusively identified while suggestions were made for the unknown components (Onayade and Adebajo, 2000;Li et al., 2009). The reversed phase column HP-5MS (5 % phenyl methyl silicone) that was used, should elute the most polar constituents first (Molnar and Horvath, 1976) and this knowledge would also assist in the identification of the resolved components.
GC-MS data obtained for AAH5 III 2-3a, AAH5 III 2-3 b and AAH5 III 4-5b were therefore utilized in the identification of the constituents of this plant being the isolates likely to contain the antimalarial constituents of the plant. These three isolates afforded 49, 8 and 55 peaks respectively. Six of the AAH5 III 2-3a peaks have > 2% peak area, the others are <2%. That of AAH5 III 2-3 b gave 4 peaks that were >3% peak area while 12 such peaks with sizes >2%. This implies that these various compounds occur in very low concentrations and it will only suffice to identify the major ones. The peaks selected eventually were the most resolved and with higher concentration in the fraction. Therefore, three, four and four major peaks were characterized respectively from the isolates using their retention times and the sizes of the peaks. Hence, the following compounds were identified as the major constituents of the plant after comparing their retention times and mass spectra data with that of standard compounds. This identified the respective compounds with retention times and peak areas as components of the resolved mixtures respectively as follows: AAH5 III 2-3a: (3 compounds) (i) ( In summary, it was observed that successive purification of the original ethanolic extract (EE) enhanced antimalarial activity at each stage; the observed antimalarial activity in the extract, partitioned and bulked column fractions being attributable to its chemical constituents. Also, previous works have shown the anti-malarial activity of chemical constituents like alkaloids and flavonoids isolated from plants (Okokon et al., 2006;Balogun et al., 2009). Also the antimalarial activity observed in this study could be due to single or combined effect of these compounds (Ebiloma et al., 2011). Although numerous bioactive agents have been isolated from the roots, stem bark and leaves of Artocarpus communis, few antimalarial studies have been reported to date (Boyom, et al., 2009). The investigation of a related species (Artocarpus integer) led to the isolation of prenylated stilbene with an IC50 of 1.7 μg/ml against Plasmodium falciparum in culture (Boonlaksiri et al., 2000).

Conclusion
In conclusion, this study has justified the use of the stem bark of Artocarpus altilis for malaria therapy in traditional medicine.